Abstracts

Characterizing Antigen Specific B cells in Thoracic Organ Transplantation

Marlena Habal, MD Assistant Professor of Medicine, NYU Grossman School of Medicine

​

Although transplantation remains the gold-standard for end-stage heart and lung disease with excellent 1- and 5-year outcomes, antibodies against the donor organ continue to limit both access to this life-saving therapy (pre-transplant sensitization) and long-term survival (chronic rejection). Despite the grave consequences, our understanding of the cellular sources of these pathogenic antibodies remains limited. Conversely, the COVID-19 pandemic highlighted the challenges of generating protective immunity in this chronically immunosuppressed population. B-cells have the capacity to retain memory, differentiate into antibody secreting cells, and serve as antigen presenting cells collectively underscoring their relevance to both rejection and infection. Novel strategies that combine antigen-baiting with multi-parameter flow cytometry, single-cell sorting, and cell culture techniques to address gaps in our understanding of humoral immunity in this population will be discussed.

​

Spectral flow cytometry: Development of high-parameter panels for the diagnosis

and treatment follow up of pediatric leukemia.

 Paul Mead, PhD, SCYM Principal Scientist, Department of Pathology, St. Jude Children's Research Hospital

​

Peixin Liu, Laura Key, Christy Embrey, Priyadarshini Kumar, Mahsa Khanlari, Jeffery M. Klco, Paul E. Mead* At St Jude Children’s Research Hospital, the current platform for clinical flow cytometry is the three-laser, eight-parameter BD CantoII cytometer. These instruments are nearing the end of their functional lifespan. Due to the complexity of the panels run on each patient sample, many separate tubes are required to cover the markers needed. For example, our current diagnostic Immunophenotyping (IP) panel for new, or relapsed, leukemias consists of ten tubes of surface markers and three tubes of cytoplasmic markers, covering more than 40 discrete antigens. Spectral flow cytometry differs from conventional cytometry by measuring the entire near-visible spectral output of a cell rather than simply counting photons in discrete regions of the spectra that coincide with the emission maxima of certain fluorochromes. This increase in the complexity of the measured spectral emission allows for many more fluorochromes to be used in a single experiment. We are in the process of migrating our high-complexity flow cytometry assays, IP and minimal residual disease (MRD) panels, to a spectral flow cytometry platform. Clinical validation of a single-tube 38-parameter spectral IP panel is underway and development of lineage-specific MRD panels is ongoing. Examples of these panels, with patient data and comparisons to the conventional CantoII analyses, will be presented.

​

Careers in Cytometry Panel Discussion

​

Flow cytometry is a booming field that has something for everyone. Running a fast-paced core facility, designing your own software or algorithm, engineering new instruments or being the go-to trouble shooter for current ones, bringing solutions to the problems of researchers and clinicians through sales, or diagnosing a patient’s illness and bringing them new treatments. This is just a small sampling of what we can do in the field of flow cytometry. Join us as we gather successful professionals from our field as they discuss their roles so we can learn more about our co-workers that we spend so much time with or you just might find out about the dream career that you didn't even know existed in Flow!

​

" 'That’s What I Do!' : A Peter Lopez Retrospective"

Peter Lopez Associate Professor of Pathology, NYU Grossman School of Medicine

​

I’m happy to have the opportunity to publicly review my journey in the field of flow cytometry and laboratory management, which created the basis of an ongoing career spanning 46 years as of 2023. In this presentation I’ll discuss my interests, what I did before flow cytometry, how I got myself into this business, my research interests and caccomplishments, and the tools that I accumulated along the way. I’ll provide my thoughts on how individuals can grow in this unique field, and my thoughts on the future.

​

Biomarkers of disease activity in Juvenile Dermatomyositis:

Focus on B cells and T cells that lack follicular markers

Preetha Balasubramanian, PhD Post-Doctoral Associate, Gale and Ira Drukier Institute for Children's Health, Weill Cornell Medical College, New York

​

Juvenile dermatomyositis (JDM) is a childhood-specific inflammatory myopathy characterized by proximal muscle weakness, unique skin manifestations and systemic vasculopathy. B and T cells are thought to contribute to JDM pathogenesis. In this study, we sought to characterize B and T-cell phenotypic characteristics, including follicular homing markers. The study comprised a total of 123 juvenile DM patients and 53 healthy controls. Clinical and laboratory phenotyping of juvenile DM patients were included in the study. Surface phenotype of peripheral blood mononuclear cells was assessed using flow cytometry. Whole blood transcriptional profiles were studied using either RNA-sequencing or microarrays. Differential gene expression was determined using DESeq and compared by pathway and gene ontology analyses. Conventional memory (CD27+IgD–) B cells expressing low CXCR5 levels (CXCR5low/– CM B cells) were significantly increased in frequency and absolute numbers in 2 independent cohorts of juvenile DM patients compared with healthy controls. The frequency of CD4+ Th2 memory cells (CD45RA–CXCR5–CCR6–CXCR3–) was also increased in juvenile DM, especially in patients who were within <1 year from diagnosis. The frequency of CXCR5low/– CM B cells correlated with serum aldolase levels and with a blood interferon-stimulated gene transcriptional signature. Furthermore, both the frequency and absolute numbers of CXCR5low/– CM B cells correlated with clinical and laboratory measures of muscle DA (MMT-8, CMAS, aldolase, and LDH). These findings suggest that both CM B cells lacking the CXCR5 follicular marker and CXCR5– Th2 cells represent potential biomarkers of DA in juvenile DM and may contribute to its pathogenesis.

​

Neuro-immune circuits control intestinal inflammation and tissue protection in the context of IBD

Hiroshi Yano, PhD Postdoctoral Associate Weill Cornell Medicine

​

Inflammatory bowel diseases (IBD) represent a growing public health and economic burden and are characterized by diarrhea, bleeding, and debilitating chronic intestinal inflammation and tissue damage. While various host intrinsic and environmental factors influence IBD pathogenesis, recent studies revealed a regulatory role for the nervous system and group 2 innate lymphoid cells (ILC2s) in controlling intestinal inflammation. However, the cellular and molecular mechanisms through which neuro-immune circuits control intestinal inflammation and tissue protection remain poorly defined. We hypothesize that neuronal-ILC2 interactions regulate intestinal inflammation and promote tissue protection in IBD. To investigate the regulatory role of neuro-immune circuits and ILC2s in IBD pathogenesis, we developed a new mouse model for specific genetic targeting of murine ILC2s and employed a mouse model of intestinal damage and inflammation coupled with analysis of human samples to analyze this. Inflamed intestinal tissues isolated from IBD patients exhibit upregulated expression of the cholinergic neuropeptide neuromedin U (NMU), which was also recapitulated in a murine model of intestinal damage and inflammation. Further, NMU was predominantly produced by enteric neurons and selectively induced the production of tissue-protective amphiregulin (AREG) in human and murine intestinal ILC2s. Therapeutic administration of NMU significantly ameliorated intestinal damage and inflammation in mice, and NMU-mediated tissue protection is dependent on ILC2-derived AREG. These observations indicate that neuronal regulation of non-redundant tissue-protective functions of ILC2s is an evolutionarily conserved mechanism that governs intestinal tissue protection. Targeting neuro-immune pathways may provide novel therapeutic intervention strategies for the treatment and prevention of IBD and associated disorders.

​

​

MSKCC Flow Cytometry Core Facility, Discussion on SRL & Research Activities

​

Fit-for-Purpose Validation Requirements for High-Parameter Cytometry Assays

Shuyu Yao, PhD Associate Director, GSK

​

Recent advancements in flow cytometry instrumentation and fluorochrome availability have resulted in significant increases in the complexity and dimensionality of flow cytometry panels. This increase in panel size allows for detection of a broader range of markers and populations, but it also comes with many challenges in assay validation and interpretation of reportable results. I will describe the practices we established for fit-for-purpose (FFP) validation of 23-color Aurora spectral flow cytometry panel used to evaluate the immune response in patients receiving engineered TCR T cells. This assay provides info on cellular kinetics and deep profiling of engineered T cells in cryopreserved PBMCs from patient samples pre and post infusion. FFP validation of this assay was performed using unstimulated and stimulated PBMCs from healthy donors and samples spiked with engineered T cell drug product. Large number of parameters were subjected to intra-assay, inter-assay, inter-instrument, and inter-operator precision assessment. During clinical testing we encountered issues with instability of critical reagent used for tracking of engineered drug product. In addition, spillover issues resulting from instability of CD3-APC-Fire810 were also observed. This resulted in assay modifications that required panel re-design and additional validations concurrent with clinical sample testing. Furthermore, we explored the use of automated analysis approaches on quality control material across different runs for batch-to-batch comparisons and assessment of staining and spectral unmixing quality. Assay validation is critical in aiding and guiding biomarker data interpretation. Better implementation of FFP validation approaches on exploratory high-parameter panels will result in generation of more robust reportables. The human biological samples used were sourced ethically, and their research use was in accord with the terms of the informed consents under an IRB/EC approved protocol.

​

​

​

​